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  • Hypoxia Signaling Cascade for Erythropoietin Production inHepatocytes

    Yutaka Tojo,a,b,c Hiroki Sekine,a,b Ikuo Hirano,b Xiaoqing Pan,a,b* Tomokazu Souma,b* Tadayuki Tsujita,b,d Shin-ichi Kawaguchi,d*Norihiko Takeda,e Kotaro Takeda,f Guo-Hua Fong,f Takashi Dan,d Masakazu Ichinose,c Toshio Miyata,d Masayuki Yamamoto,b

    Norio Suzukia

    Division of Interdisciplinary Medical Science,a Department of Medical Biochemistry,b Department of Respiratory Medicine,c and Division of Molecular Medicine andTherapy,d Tohoku University Graduate School of Medicine, Sendai, Japan; Department of Cardiovascular Medicine, Graduate School of Medicine, University of Tokyo,Tokyo, Japane; Center for Vascular Biology, Department of Cell Biology, University of Connecticut Health Center, Farmington, Connecticut, USAf

    Erythropoietin (Epo) is produced in the kidney and liver in a hypoxia-inducible manner via the activation of hypoxia-inducibletranscription factors (HIFs) to maintain oxygen homeostasis. Accelerating Epo production in hepatocytes is one plausible thera-peutic strategy for treating anemia caused by kidney diseases. To elucidate the regulatory mechanisms of hepatic Epo produc-tion, we analyzed mouse lines harboring liver-specific deletions of genes encoding HIF-prolyl-hydroxylase isoforms (PHD1,PHD2, and PHD3) that mediate the inactivation of HIF1 and HIF2 under normal oxygen conditions. The loss of all PHD iso-forms results in both polycythemia, which is caused by Epo overproduction, and fatty livers. We found that deleting any combi-nation of two PHD isoforms induces polycythemia without steatosis complications, whereas the deletion of a single isoform in-duces no apparent phenotype. Polycythemia is prevented by the loss of either HIF2 or the hepatocyte-specific Epo geneenhancer (EpoHE). Chromatin analyses show that the histones around EpoHE dissociate from the nucleosome structure afterHIF2 activation. HIF2 also induces the expression of HIF3, which is involved in the attenuation of Epo production. Theseresults demonstrate that the total amount of PHD activity is more important than the specific function of each isoform for he-patic Epo expression regulated by a PHD-HIF2-EpoHE cascade in vivo.

    Under low-oxygen conditions (hypoxia), the number of circu-lating red blood cells increases to deliver oxygen efficientlyinto peripheral organs. The red blood cell mass is controlled by theerythroid growth factor erythropoietin (Epo), the majority ofwhich is produced by the kidneys in a hypoxia-dependent manner(1). Therefore, inhabitants of high-altitude areas and patients suf-fering from chronic respiratory failure with chronic obstructivepulmonary disease often develop polycythemia (erythrocytosis)with elevated levels of Epo in their plasma (2, 3). Epo binds to itsreceptor (EpoR) on the surfaces of immature erythroid cells andstimulates signaling cascades for proliferation, differentiation,and antiapoptosis (4). Epo or EpoR gene-targeted mouse lines areembryonic lethal, with severe anemia around embryonic day 13,clearly indicating a requirement for Epo signaling in erythropoi-esis (5).

    Anemia often occurs in patients who suffer from kidney dam-age (6). We previously reported that renal Epo-producing (REP)cells that are in the interstitial spaces between renal tubules trans-form into myofibroblastic cells, which are closely associated withrenal fibrosis under inflammatory conditions (711). Because thetransformed REP cells lose their ability to produce Epo even underseverely hypoxic conditions, pharmacologically inducing Epoproduction in the damaged kidneys of anemic patients is difficult.Although REP cells secrete most of the Epo in adult animals, hepa-tocytes are the primary Epo-producing cells in fetuses (12). Theliver maintains its Epo-producing activity throughout adulthood;thus, Epo expression is detectable in the livers of anemic/hypoxicmice (8, 12). However, the level of hepatic Epo production is weakand insufficient to compensate for renal anemia (13). Therefore,pharmacological enhancement of hepatic Epo production is a rea-sonable strategy for treating anemic patients who have renal dis-eases (14, 15).

    In hepatocytes, Epo transcription, which is the rate-limitingstep of Epo production, is controlled by a cis-regulatory elementthat is proximally downstream of the Epo transcription end site(called Epo hepatic enhancer [EpoHE] in this study). EpoHE con-tains a binding sequence for hypoxia-inducible transcription fac-tors (HIFs) (16). We previously demonstrated that EpoHE is nec-essary and sufficient for Epo expression in hepatocytes, whereasrenal Epo production is independent of EpoHE (12, 13). Thus,while Epo expression is commonly induced by hypoxic stresses inREP cells and hepatocytes, these two cell types employ differentmechanisms of Epo gene regulation.

    HIFs are master regulators of hypoxia-inducible gene expres-sion. Each HIF consists of an oxygen-responsive subunit and aconstitutively expressed nuclear subunit (17). Mammalian ge-

    Received 11 February 2015 Returned for modification 16 March 2015Accepted 22 May 2015

    Accepted manuscript posted online 26 May 2015

    Citation Tojo Y, Sekine H, Hirano I, Pan X, Souma T, Tsujita T, Kawaguchi S-I,Takeda N, Takeda K, Fong G-H, Dan T, Ichinose M, Miyata T, Yamamoto M, SuzukiN. 2015. Hypoxia signaling cascade for erythropoietin production in hepatocytes.Mol Cell Biol 35:2658 2672. doi:10.1128/MCB.00161-15.

    Address correspondence to Norio Suzuki, sunorio@med.tohoku.ac.jp.

    * Present address: Xiaoqing Pan, Division of Rare Cancer Research, National CancerCenter Research Institute, Tokyo, Japan; Tomokazu Souma, Division ofNephrology, Northwestern University Feinberg School of Medicine, Chicago,Illinois, USA; Shin-ichi Kawaguchi, Department of Applied Chemistry, GraduateSchool of Engineering, Osaka Prefecture University, Osaka, Japan.

    Y.T. and H.S. contributed equally to this work.

    Copyright 2015, American Society for Microbiology. All Rights Reserved.

    doi:10.1128/MCB.00161-15

    2658 mcb.asm.org August 2015 Volume 35 Number 15Molecular and Cellular Biology

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  • nomes contain three genes for HIF isoforms. HIF1 and HIF2have similar protein structures, and both exhibit strong transac-tivities under hypoxic conditions, whereas the function and regu-latory mechanisms of HIF3 remain controversial due to the ex-istence of multiple HIF3 splicing variants (1820). Undernormal oxygenation conditions (normoxia), HIF subunits areconsistently synthesized, and their specific proline residues arequickly hydroxylated by HIF-prolyl hydroxylase domain proteins(PHDs) (21, 22). Hydroxylated HIF is recognized by von Hip-pel-Lindau protein (pVHL), which is a component of an E3 ubiq-uitin ligase complex, and degraded by the proteasome (23). Be-cause hypoxia inhibits the enzymatic activities of PHDs, which useoxygen as a substrate, HIF heterodimers have increased activitiesas transcription factors under hypoxic conditions (24).

    Hepatic Epo production is enhanced by the experimental tar-geting of the HIF pathway using genetically modified mice, smallmolecules, or small interfering RNA s (siRNAs) (25). For instance,liver-specific pVHL deficiency in mice results in polycythemiawith the overexpression of the Epo gene in the liver (26). Becausethis phenotype depends on HIF2, it is thought that HIF2,rather than HIF1, regulates hypoxia-inducible Epo expression inhepatocytes (26). The contributions of the three PHD isoforms(PHD1, PHD2 and PHD3, which are encoded by the Egln2, Egln1,and Egln3 genes, respectively) to hepatic Epo gene regulation havebeen investigated using gene-modified mice, and functional re-dundancy among the isoforms has been demonstrated (2729).Although these strategies stimulate hepatic Epo production, theconstitutive activation of HIFs induces steatosis and ectopic ex-pression of cancer-related genes (2529). Therefore, establishing aspecific strategy for hepatic Epo induction to treat renal anemia isimportant, and fully understanding the molecular mechanismunderlying Epo regulation will be necessary to develop such astrategy.

    In this study, we analyzed gene-modified mouse lines lackingPHD isoforms, HIF2, and/or EpoHE specifically in the liver to elu-cidate the in vivo signaling pathway between hypoxia sensor PHDsand cis-regulatory elements in the hepatic Epo gene regulatory sys-tem. Our results demonstrate that the function of PHDs is gene dosedependent and that HIF2 and EpoHE are necessary for hepatic Epoproduction. HIF2 functions in both the removal of histones fromthe region around the Epo gene, which enhances Epo transcription,and the induction of HIF3 expression to attenuate hepatic Epo pro-duction. Thus, we propose that a PHD-HIF2-EpoHE axis regulateshepatic Epo production by controlling HIF3 expression and thechromatin structure of the Epo gene. Our results provide valuableinformation regarding therapeutic targets that enhance hepatic Epoproduction for renal-anemia patients.

    MATERIALS AND METHODSMice. All mice were maintained at the Institute for Animal Experimenta-tion, Tohoku University Graduate School of Medicine, under the Regu-lations for Animal Experiments and Related Activities of Tohoku Univer-sity. A hypoxic box, in which the oxygen concentration was regulated byan oxygen controller (ProOx; BioSpherix) with a nitrogen generator(Nilox; Sanyo Electronic Industries), was used to expose the mice to hy-poxic conditions. For acclimation to severe hypoxia (6% O2), the micewere exposed to 10% oxygen for 2 h before the oxygen concentration wasreduced to 6%. Mouse lines carrying conditional knockout genes forPHDs (Egln1f/f, Egln2f/f, and Egln3f/f genoty

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